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In the latter case, a wide range of methods have been developed starting from the first strategy named single-specific-primer PCR [9]. These methods are cata-logued according to Tonooka and Fujishima [3] as ‘cassette PCR’, for the use of double-stranded DNA linkers to ligate to genomic DNA restriction frag-ments. We prefer the term ‘cassette PCR’ instead of ’ligation mediated PCR’, which is sometimes also used to indicate these methods ([7,21,22,28,44] 更多：https://www.bmcx.com/ , for exam-ple), since the term ‘ligation mediated’ has been used since 1989 to indicate one of the first GW strategies, here classified among the E-GW methods (Table 1). Once the cassette has been ligated to genomic DNA restriction fragments, generating what is commonly known as the GW library, a PCR amplification of the region encompassing the boundary between the known and unknown sequences can be carried out using a sequence specific primer and a cassette specific primer. One major concern in the cassette PCR based methods is the background of non-specific products due to the cassette specific primer. To overcome this problem a number of tricks have been devised, such as those adopted in vectorette PCR [11], capture PCR [13] and other strategies reported below.